Methods and compositions for dual glycan binding AAV vectors

Abstract

The present invention provides methods and compositions comprising an adeno-associated virus (AAV) capsid protein, comprising one or more amino acids substitutions, wherein the substitutions introduce a new glycan binding site into the AAV capsid protein.

That which is claimed is: 
     
       1. A modified adeno-associated virus (AAV) capsid protein, comprising amino acid substitutions that result in the amino acid sequence:
 a) SVAGPSNMAVQGR at positions corresponding to amino acids 464-476 of AAV9 (SEQ ID NO:75); and 
 b) EFAW at positions corresponding to amino acids 500-503 of AAV9 (SEQ ID NO:75); 
 
       wherein the amino acid substitutions introduce a galactose binding site from donor AAV9 capsid protein into the modified AAV capsid protein. 
     
     
       2. The modified AAV capsid protein of  claim 1 , wherein the modified AAV capsid protein is selected from the group consisting of human AAV and non-human AAV. 
     
     
       3. The modified AAV capsid protein of  claim 2 , wherein the non-human AAV is selected from the group consisting of non-human primate AAV, avian AAV, and bovine AAV. 
     
     
       4. The modified AAV capsid protein of  claim 1 , wherein the serotype of the modified AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, Avian AAV, BB1, BB2, CHS, CY2, CY3, CY4, CY5, CY6, Hu LG15, Hu S17, Hu T17, Hu T41, Hu T70, Hu T71, Hu T88, Hu1, Hu2, Hu3, Hu4, Hub, Hu7, Hu9, Hu10, Hu11, Hu13, Hu15, Hu16, Hu17, Hu18, Hu19, Hu20, Hu21, Hu22, Hu23, Hu24, Hu25, Hu27, Hu28, Hu29, Hu34, Hu35, Hu37, Hu39, Hu40, Hu41, Hu42, Hu43, Hu44, Hu45, Hu46, Hu47, Hu48, Hu49, Hu51, Hu52, Hu54, Hu55, Hu56, Hu57, Hu58, Hu60, Hu61, Hu63, Hu64, Hu66, Hu67, Rh1, Rh2, Rh8, Rh10, Rh12, Rh13, Rh14, Rh16, Rh17, Rh18, Rh19, Rh22, Rh23, Rh24, Rh25, Rh26, Rh27, Rh31, Rh32, Rh33, Rh34, Rh35, Rh36, Rh37, Rh38, Rh40, Rh43, Rh48, Rh49, Rh50, Rh51, Rh52, Rh53, Rh54, Rh55, Rh57, Rh58, Rh60, Rh61, Rh62, and Rh64. 
     
     
       5. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV10. 
     
     
       6. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV1 and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:67, are G466A, S467G, A469S, G470N, S472A, P475G, K476R, N500E, and T502A. 
     
     
       7. The modified AAV capsid protein of  claim 1 , wherein the serotype of the modified AAV capsid protein is AAV2. 
     
     
       8. The modified AAV capsid protein of  claim 7 , further comprising the following amino acid substitutions: R585Q, G586Q, R588T, Q589A, and A590P. 
     
     
       9. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV3a and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:68, are Q465V, Q469S, S470N, S472A, L473V, A475G, N500E, and P502A. 
     
     
       10. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV3b and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:69, are Q465V, Q469S, S470N, S472A, L473V, A475G, N500E, and P502A. 
     
     
       11. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV4 and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO: 70, are T458S, K459V, L460A, R461G, T463S, F465M, S466A, N467V, F468Q, K469G, K470R, D499E, S500F, L501A, and I502W and further wherein the amino acids S, S, N and D are inserted as a contiguous sequence immediately before the amino acid N at position 261. 
     
     
       12. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV5 and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:71, are N450S, K451V, N452A, L453G, A454P, G455S, R456N, Y457M, N459V, T460Q, Y461G, K462R, V486E, S487F, and F489W. 
     
     
       13. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV6 and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:72, are R465V, G466A, S467G, A469S, G470N, S472A, P475G, K476R, N500E, and T502A. 
     
     
       14. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV7 and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:73, are Y466S, Q467V, G468A, T472N, E475V, A477G, K478R, and N502E. 
     
     
       15. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV8 and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:66, are Q467V, G468A, N471S, T472N, N475V, A477G, K478R, and N502E. 
     
     
       16. The modified AAV capsid protein of  claim 4 , wherein the serotype of the modified AAV capsid protein is AAV10 and the corresponding amino acid substitutions, based on the amino acid sequence of SEQ ID NO:74, are Q467V, A471S, S474A, A475V, A477G, K478R, and N502E. 
     
     
       17. An AAV capsid comprising the modified AAV capsid protein of  claim 1 . 
     
     
       18. A virus vector comprising:
 (a) the AAV capsid of  claim 17 ; and 
 (b) a nucleic acid comprising at least one terminal repeat sequence, 
 
       wherein the nucleic acid is encapsidated by the AAV capsid. 
     
     
       19. A composition comprising the modified AAV capsid protein of  claim 1  in a pharmaceutically acceptable carrier. 
     
     
       20. A composition comprising the AAV capsid of  claim 17  in a pharmaceutically acceptable carrier. 
     
     
       21. A composition comprising the virus vector of  claim 18  in a pharmaceutically acceptable carrier. 
     
     
       22. A method of introducing a nucleic acid into a cell, comprising contacting the cell with the virus vector of  claim 18 . 
     
     
       23. The method of  claim 22 , wherein the cell is in a subject. 
     
     
       24. The method of  claim 23 , wherein the subject is a human subject. 
     
     
       25. The modified AAV capsid protein of  claim 1 , further comprising an amino acid substitution that results in a S at position corresponding to amino acid 268 of AAV9 (SEQ ID NO:75).